The joining, or ligation, of DNA molecules is an essential step in replication, recombination, and repair of the genetic material of all living organisms. Therefore, it is not surprising that organisms ranging from viruses to humans have evolved an enzymatic system to take care of this important metabolic reaction. This enzymatic system is designated as DNA LIGASE, EC 6.5.1.1. (1-5).
Over the past four decades, considerable attention has been devoted to the study of the biochemistry and molecular biology of DNA ligases from a variety of organisms (for reviews, see 6-10). These studies have clearly established the importance of DNA ligases in maintaining the integrity of genetic material. Recent results also indicate a correlation between lack of DNA ligase activity and certain pathological conditions in humans (11-18). Furthermore, DNA ligases have also been used as invaluable tools for advancing basic research via recombinant DNA technology (19-22). Finally, DNA ligases, in concert with the polymerase chain reaction (PCR) technology, are also being used as important diagnostic reagents (23-28).
Consequently, over the years, DNA ligases have been purified and characterized from a variety of sources (2-5). Different methods have also been developed for assaying this group of enzymes. These assay methods can be broadly divided into two groups:
I. Enzvme-intermediate Characterization:
Assays involving enzyme-intermediate characterization take advantage of the fact that the DNA ligases require ATP (for viral and eukaryotic enzymes) or NAD (for bacterial enzymes) as a cofactor. The co-factor forms an adenylylated intermediate with the enzyme. Use of radiolabeled co-factor then allows one to follow the presence of DNA ligase-like activity in crude extracts (7). Such assays can be performed relatively easily and may serve as qualitative tests to indicate the presence or absence of ligase-like polypeptides in crude extracts. However, this method has several drawbacks. First of all, it is not specific for DNA ligases only. RNA ligases (29, 30) as well as mRNA capping enzymes (31) will also form AMP-adducts, and thereby can complicate interpretation of results. Secondly, this method uses radioactivity, necessitating additional precautions and waste disposal costs. Finally, this method does not constitute a true biological assay, and therefore requires additional characterization of the substrate/product for confirmation.
II. Substrate/Product Characterization:
These assays may be further divided into two subgroups: (a) structural and (b) functional. Most of the methods published thus far involve structural characterization of the substrate and/or product (7, 26, 32). Thus, two double-stranded DNA fragments, one with a free 3' --OH group and the other with a 5'--PO.sub.4 group are incubated with the enzyme under appropriate conditions. The disappearance of the substrate or appearance of the products can be detected by electrophoresis alone, or by electrophoresis in combination with autoradiography (if the substrates are radiolabeled). Another variation of this method is to measure the incorporation of .sup.32 P--PO.sub.4 into the phosphatase-resistant product, thereby making quantitation easier. Although these methods are currently used routinely, they also suffer from the use of radioactivity and require additional characterization of the product to prove functionality.
Recently, use .of fluorescent labels for DNA substrates has been shown to have the potential to replace use of radioactivity (27, 28), thereby reducing the health related concerns, as well as increasing sensitivity significantly. However, use of sophisticated instruments makes these methods less cost effective. Moreover, the biological function of the product needs to be established separately when these methods are used. This is routinely achieved by using a plasmid containing a selectable marker (e.g. antibiotic resistance) as a substrate for the enzymatic reaction (7). Upon ligation, the reaction product is used to transform bacteria (e.g. E. coli) that are grown in presence of the specific antibiotic. Only those bacteria which harbor the ligated plasmid DNA containing a functionally reconstituted antibiotic marker gene will grow, indicating successful ligation and restoration of function of the product. Hence, this assay qualifies as a functional biological assay (7). However, this biological assay for DNA ligase activity (believed to be the only such biological assay currently available) is time consuming (at least two days), laborious, and qualitative rather than quantitative. Therefore, there is a particular need for a functional, quantitative assay for DNA ligase activity. The present invention describes a functional assay that is non-radioactive, extremely sensitive, quantitative, and that can be completed within 3.5 hours. Furthermore, this assay can be applied to all types of DNA ligases.